Uses of hydroxyl polymethoxylflavones (HPMFs) and derivatives thereof

ABSTRACT

The invention discloses the uses of hydroxyl polymethoxylflavones and derivative thereof that are relative to inhibit adipogenesis and lower lipid accumulation, wherein the hydroxyl polymethoxylflavones is obtained from the peels of  Citrus  genus plants. Therefore, the hydroxyl polymethoxylflavones that of a therapeutically effective amount not only can be a medical compound for treatment obesity or fatty liver, but also can be a food element.

CROSS-REFERENCE TO RELATED APPLICATION

The current application claims a foreign priority to the patentapplication of Taiwan No. 102129499 filed on Aug. 16, 2013.

FIELD OF THE INVENTION

The present invention relates generally to the uses of the naturecompound. In particular, the present invention relates to uses ofhydroxyl polymethoxylflavones (HPMFs) and derivatives thereof.

BACKGROUND OF THE INVENTION

According to the previous reports, obesity is resulted from thatassimilated calorie is much than consumption. The excessive calorie isstored as triglyceride (hereinafter referred to as TG) in adipose tissueand thereby accumulated in pancreas, liver, skeleton muscle. Theaccumulation of triglyceride in these indicated organs causes obesityand metabolic syndrome such as cardiovascular diseases, hypertension,hyperlipidemia and type II diabetes. Currently, WHO has been categorizedobesity as a chronic illness disease. In Taiwan, the nutritional healthreports from 2004 to 2008 had suggested that the excessive nutritionwould cause the occurrence of many chronic illness diseases.

The obesity could be cured by various therapy approaches such asexercise, calorie control and surgery. However, eating out is the commondietary habit of people in the rush modern world. Therefore, the rushmodern life style makes it difficult to low body weight through exerciseand calorie control. Furthermore, the cost of the surgeries for lowingbody weight such as cosmetic surgery and laparoscopic gastric bypasssurgery are quite expansive. In addition to the expansive cost, thesurgeries may bring some uncertain risk for the safety of patients. Somepharmaceutical approaches such as metabolic stimulus, appetite inhibitorand starch blocker are approached to low the body weight. However,intake of these drugs for a long term will bring some side effects tothe subjects. In order to avoid the side effects of these availabledrugs, many investigations have suggested that the nature compounds andextracts are capable of lowing body weight through inhibiting appetiteor accelerating the metabolism of energy. For example, caffeineaccelerates energy metabolism through inhibition of phosphodiesteraseactivity to increase the cyclic adenosine monophosphate. The extract ofHoodia Cactus is capable of suppressing the appetite; the extract ofPrunella vulgaris is able to be the starch blocker through inhibitingthe enzyme activity of salivary amylase; the extract of Semenplantaginis is capable of lowing the concentration of TG in blood byaccelerating the lipid metabolism.

Because these indicated natural extracts do not achieve the purpose forlowing weight through controlling the cell cycle of adipocytes,inhibiting adipocytes differentiation, promoting the lipolysis inadipocytes, the functions of these extracts are not efficient andobvious. Therefore, the current investigations have focused ondevelopment of new natural compounds for controlling behavior ofadipocytes or suppressing the lipogenesis to efficiently suppress thelipid accumulation and obesity.

SUMMARY OF THE INVENTION

The present invention relates a hydroxyl polymethoxylflavones compound,hereinafter referred to as a HPMFs compound, which is extracted fromcitrus fruits. The HPMFs compound has ability for inhibition oflipogenesis and adipocyte differentiation, and decreasing the lipidaccumulation surrounding the internal organs without alteration on therate of food intake. Therefore, the HPMFs compound or derivative thereofcan serve as an active ingredient of a composition for treating orpreventing obesity-related diseases.

It is an object of the present invention provides a method of treatingor preventing obesity-related diseases comprising administering to asubject a composition in a effective amount, wherein the compositionincluding a HPMFs compound or a pharmaceutically acceptable saltthereof, or mixture thereof.

According to the present invention, the HPMFs compound is extracted froma peel of citrus fruit, wherein, the citrus fruit would be pomelo,mandarin orange, orange, kumquat or lemon.

In one embodiment of the present invention, the HPMFs compound isextracted by the following steps: (A) taking a predetermined amountextract from the peel of citrus fruit; (B) dissolving the extract withalcohols and adding hydrochloric acid to obtain a mixture; (C)incubating the mixture with heating circumfluence and then removingalcohols from the mixture; (D) extracting the mixture of the step C bywater and an organic solvent; (E) collecting the organic solvent phaseand purifying to obtain the HPMFs compound.

According to the present invention, the obesity-related disease isincluding, but not limiting to, obesity, fatty liver, metabolicsyndrome, insulin resistance syndrome, cardiovascular disease,hypertension and hyperlipidemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the cellular morphology of cultured 3T3-L1 pre-adipocyteson day 0.

FIG. 1B shows the cellular morphology of cultured 3T3-L1 pre-adipocyteson day 2.

FIG. 1C shows the cellular morphology of cultured 3T3-L1 pre-adipocyteson day 4.

FIG. 1D shows the cellular morphology of cultured 3T3-L1 pre-adipocyteson day 6.

FIG. 1E shows the cellular morphology of cultured 3T3-L1 pre-adipocyteson day 8.

FIG. 1F shows the oil red staining of cultured 3T3-L1 pre-adipocytes onday 8.

FIG. 2A˜2B show the oil red staining of cultured 3T3-L1 pre-adipocyteswith different treatments on day 8 after differentiation.

FIG. 2C shows the content of triglyceride in cultured 3T3-L1pre-adipocytes with different treatments on day 8 after differentiation.

FIG. 3A shows the expression level of PPARγ in cultured 3T3-L1pre-adipocytes with different treatments on day 8 after differentiation.

FIG. 3B shows the expression level of C/EBPα in cultured 3T3-L1pre-adipocytes with different treatments on day 8 after differentiation.

FIG. 4A shows the expression level of fatty acid synthase in cultured3T3-L1 pre-adipocytes with different treatments on day 8 afterdifferentiation.

FIG. 4B shows the expression level of fatty acid binding protein 2 (aP2)in cultured 3T3-L1 pre-adipocytes with different treatments on day 8after differentiation.

FIG. 4C shows the expression level of acetyl-coA carboxylase in cultured3T3-L1 pre-adipocytes with different treatments on day 8 afterdifferentiation.

FIG. 5A shows the expression levels of AMP-activated protein kinase-α(AMPK-α) and phospho-AMPK-α (Thr172) in cultured 3T3-L1 pre-adipocyteswith different treatments on day 8 after differentiation.

FIG. 5B shows the expression levels of AMP-activated protein kinase-β(AMPKβ) and phospho-AMPKβ (Ser108) in cultured 3T3-L1 pre-adipocyteswith different treatments on day 8 after differentiation.

FIG. 5C shows the expression level of sterol regulatory element bindingprotein 1c (SREBP 1c) in cultured 3T3-L1 pre-adipocytes with differenttreatments on day 8 after differentiation.

FIG. 6 shows the expression levels of PI3K, phospho-PI3K (Tyr508), AKTand phospho-AKT (Ser473) in cultured 3T3-L1 pre-adipocytes withdifferent treatments on day 8 after differentiation.

FIG. 7A˜7B shows the cell cycle analysis of cultured 3T3-L1pre-adipocytes with different treatments.

FIG. 8 shows the changes in the body weights of the each group mice withdifferent administrations.

FIG. 9 shows the dietary intakes of the each group mice with differentadministrations.

FIG. 10A˜10D show the gross views of the each group mice with differentadministrations.

FIG. 11A˜11D show the gross views of the fat pads surrounding gonad ofthe each group mice with different administrations.

FIG. 12A˜12D show the gross views of the abdominal fat pads of the eachgroup mice with different administrations.

FIG. 13 shows the weights of the fat pads surrounding the gonad of theeach group mice with different administrations.

FIG. 14 shows the weights of the abdominal fat pads of the each groupmice with different administrations.

FIG. 15 shows the fat pads surrounding intestine of the mice in the eachgroup with different administrations.

FIG. 16A˜16D show H&E staining for hepatic histology of the each groupmice with different administrations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a HPMFs compound for inhibitinglipogenesis, suppressing adipocyte differentiation and preventing lipidaccumulation. The HPMFs compound and/or derivatives thereof with aneffective dosage could be used as an active ingredient of a compositionfor treating or preventing obesity-related diseases. Moreover, the HPMFscompound is extracted from a peel of genus citrus. By administering to asubject the composition, it is helpful for suppression of adipocytedifferentiation and decrease of adipocyte accumulation.

Furthermore, the HPMFs compound is generated from polymethoxylflavones(PMFs) by replacing at least one methoxyl- group (—OCH3) by hydroxyl-group (—OH).

The previous studies have indicated that differentiation process from3T3-L1 pre-adipocytes to the mature adipocytes is mediated by varioussignaling pathways. For example, the lipogenesis promoting transcriptionfactors such as PPAR and C/EBPs are capable of triggering the maturationof 3T3-L1 pre-adipocytes. Herein, c/EBPs can activate the downstreamtarget that involves in lipid metabolism such as fatty acid bindingprotein 2 (hereinafter referred to as aP2). In addition, SREBP-1 is thecritical factor for cell fate determination of adipocyte differentiationthrough activating downstream targets involving in lipogenesis such asfatty acid synthase (hereinafter referred to as FAS) and acetyl-CoAcarboxylase (hereinafter referred to as ACC). Therefore, activation ofthe target of SREBP-1 is capable of accelerating the synthesis of fattyacid and lipid, and activation of PPARγ. Moreover, LKB1/STK11 activatesAMPKα through phosphorylation on Thr172 of AMPKα to further suppress theactivity of ACC and inhibit lipogenesis. Therefore, regulation ofAdenosine monophosphate-activated protein kinase (hereinafter referredto as AMPK) is the critical indicator for anti-lipogenesis.

The HPMFs compound is capable of controlling the expressions oftranscription factors and the related signaling pathways thatparticipate in regulation of adipocyte differentiation. Therefore, theHPMFs can suppress differentiation and maturation of adipocytes. Indetail, the HPMFs compound is capable of suppressing the criticaltranscription factors for adipocyte differentiation such as PPARγ andC/EBPα. Furthermore, the HPMFs compound can inhibit the proteinsynthesis of the downstream targets such as ACC, FAS and aP2 to slowdown the accumulation of triglyceride. In addition, activation of AMPKpathway and suppression of PI3K/AKT pathway are capable of inhibitingthe adipocyte differentiation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings commonly understood by one of ordinary skill inthe art. As used in this application, including claims andspecification, the following words is only exemplary and illustrative,not limiting in scope.

The term of composition is including, but not limiting to, apharmaceutical composition and a dietary supplement, wherein thepharmaceutical composition comprises an active ingredient and one ormore pharmaceutically acceptable carriers or excipients. According theinvention, the active ingredient is the HPMFs compound or apharmaceutically acceptable salt thereof, or mixture thereof. Moreover,the pharmaceutical composition is in the form of granular, powder,pastille, capsule, suppository, liquid, suspension, drop or the solventfor injection. The dietary supplement is a functional food in the formof granular, powder, solid substance, liquid, suspension or semi-solidsubstance.

The term of pharmaceutically acceptable salt refers to the salt or freeform of the acidic or basic product produced from the HPMFs, wherein theHPMFs and stereoisomers thereof are both capable of forming the salt.

The term of effective dose refers to the amount or dose of a compound ora composition that is sufficient to produce an effective response uponadministration to a mammal by calculation according to the races, bodyweight and delivery methods.

The term of administering refers to a delivery method that includes oralintake, breath intake, colon absorption, epidermal absorption,subcutaneous injection, artery injection, venous injection orintra-peritoneal injection.

The term of a or an refers to one or more than one.

To understand the purposes and advantages of the present invention, itis described by the following merely examples taken in conjunction withthe accompanying drawings. It will be understood that it is not intendedto limit the invention to the described embodiments.

Example 1 Preparation of the HPMFs Compound

10 grams of orange peel extract that contained HPMFs for more than 40%was dissolved in 95% ethanol and added with 3M of hydrochloric acid(HCl). The indicated mixture was incubated with heating circumfluenceand monitored by using TLC and LC/MS for 12 hours. Until the reactionfinished and cooled, ethanol was removed by vacuum device. In thefollowing step, ethyl acetate and water were added for separation andextraction. An organic solvent phase of the extract was collected,first. The remained aqueous phase was further extract by acetyl acetateagain for isolating the organic solvent phase.

The organic solvent phases were pulled together, diluted with sodiumbicarbonate solution, water and 30% NaCl solution for wash, and thendehydrated with sodium sulfate. After filtration, decompressionconcentration and lyophilized, the gained pale yellow solid substance isa HPMFs compound.

Example 2 Culture of 3T3-L1 Pre-Adipocytes

The 3T3-L1 pre-adipocytes were cultured in 10 cm culture dish with DMEMmedium, which contains 10% fetal bovine serum (FBS), 10000 unit/mL ofpenicillium and 10000 μg/mL streptomycin, at 37° C., 5% CO2, incubator.When the growing cells occupied 70%-80% area of the cultured dish,depletion of cultured medium was followed by PBS washing. The culturedcells were added trypsin-EDTA at 37° C. After the enzymatic digestion,the cultured cells were disassociated from the dish by gentle beats andthen adding fresh FBS-containing medium to terminate the enzymaticactivity of trypsin-EDTA. The disassociated cells were further wellseparated by pipetting using pipetman several times. The cells wereevenly distributed in the culture dishes and then incubated at 37° C.,5% CO2 incubator.

Example 3 Differentiation Test of 3T3-L1 Pre-Adipocytes

The 3T3-L1 pre-adipocytes seeded into 24-wells plate were cultured inFBS-containing DMEM medium for 3 days. In the following step, theoriginal medium was replaced by another fresh FBS-containing medium for2 days extension culture. The day after 2 days extension culture isdestined as the “day 0”.

On day 0, the 3T3-L1 pre-adipocytes cultured with different conditionswere treated with DMI inductance (DEX+MIX+insulin) for 2 days (day 2) toinduce the adipocyte differentiation. After the induction of adipocytedifferentiation, the induction medium was replaced by a fresh DMEMmedium that contains 10% FBS and 5 μg/mL INS for at least 2 days culture(day 4). On day 4, day 6 and day 8, the culture medium was furtherreplaced by the new 10% FBS-containing DMEM medium.

During the culture from day 0 to day 8, the morphology of the 3T3-L1pre-adipocytes was observed every 2 days and the results were shown inFIG. 1A-1E. In addition, the Oil-red O staining of the 3T3-L1pre-adipocyte on day 8 was shown in FIG. 1F. The red spots in thestaining indicate the lipid-drops in adipocyte. The results in FIG. 1show that the morphology of the undifferentiated 3T3-L1 pre-adipocyte isspindle shape. On day 2, some cultured 3T3-L1 pre-adipocytes switchtheir morphology into round shape. On day 4, some small lipid-drops areappeared in the cultured 3T3-L1 pre-adipocytes. On day 6, theaccumulated lipid-drops in the cultured cells are increased. On day 8,the cultured 3T3-L1 pre-adipocyte differentiate into the matureadipocytes that are present with accumulation of the lipid-drops.

Example 4 Analysis of Lipid Accumulation in 3T3-L1 Pre-Adipocyte

The cultured 3T3-L1 pre-adipocytes on day 8 prepared in example 3 weredivided into 4 groups and cultured with different conditions. Herein,the group 1 was the blank control treated without additional factors,the group 2 was treated with DMI containing medium on day 2 as thecontrol group, the group 3 was treated not only MDI on day 2, but alsoadded with 10 μg/mL the HPMFs compound on day 0, day 2, day 4, and day 6and the group 4 was treated not only MDI on day 2, but also added with20 μg/mL the HPMFs compound on day 0, day 2, day 4, and day 6.

After the complete culture on day 8, the each group stained by Oil Red Ostaining was observed and recorded by photograph under microscope. Themorphology of the Oil Red O stained cells of the each group was shown inFIGS. 2A and 2B. Furthermore, the lipid content of the each group wasmeasured by spectrophotometer 510 nm and shown in FIG. 2C.

According to FIG. 2, the group 1 is the un-differentiated 3T3-L1pre-adipocytes and the morphology thereof are spindle shape. In thegroup 2, the 3T3-L1 pre-adipocytes differentiate into mature adipocytebearing round shape and have the red lipid-drops therein. In contrast tothe group 2, the 3T3-L1 pre-adipocytes in the group 3 and 4 revealextremely less red lipid-drops, respectively. The results indicate thefunction of the HPMFs compound in suppressing the lipogenesis induced byDMI-induced 3T3-L1 pre-adipocytes. Therefore, it shows that treatment ofthe HPMFs compound on the 3T3-L1 pre-adipocytes reduces the lipidcontent and significantly inhibits the lipid accumulation in the cells.

Example 5 Analysis the Mechanism of the HPMFs Compound InhibitingLipogenesis

The cultured 3T3-L1 pre-adipocytes on day 8 prepared in example 3 weredivided into 4 groups and cultured with different conditions. Herein,the group 1 was the blank control, the group 2 was treated withDMI-containing medium on day 2 as the control group, the group 3 wasadded not only MDI on day 2 but also 10 μg/mL HPMFs on day 0, day 2, day4, and day 6, respectively and the group 4 was added not only MDI on day2 but also 20 μg/mL HPMFs on day 0, day 2, day 4, and day 6.

The cell lysate of the each group was collected to determine the proteinexpression by western blot analysis. The results were present in FIG. 3to FIG. 5, wherein FIG. 3A shows the expression of PPARγ in the eachgroup, FIG. 3B shows the expression of C/EBPα in the each group, FIG. 4Ashows the expression of FAS in the each group, FIG. 4B shows theexpression of aP2 in the each group, FIG. 4C shows the expression of ACCin the each group, FIG. 5A shows the expression of AMPKα and p-AMPKα(Thr172) in the each group, FIG. 5B shows the expressions of AMPKβ andp-AMPKβ (Ser108) in the each group and FIG. 5C shows the expression ofSREBP-1c in the each group.

Furthermore, the cultured 3T3-L1 pre-adipocytes on day 8 prepared inexample 3 were divided into three groups, wherein the group 1 was theblank control, the group 2 treated with DMI on day 2 was positivecontrol and the group 3 was treated with DMI on day 2 and administratedwith 20 μg/mL HPMFs on day 0, day 2, day 4, and day 6. After theinduction culture, the cells of the each group were collected and lysedfor analysis of gene expression by western blot. The results were shownin FIG. 6 that includes the expressions of PI3K, p-PI3K (Tyr508), AKTand p-Akt (Ser473) in the each groups.

According to FIG. 3 and FIG. 4, it suggests that the expression levelsof PPARγ, C/EBPα, ACC, FAS and aP2 in the group 2 are increased withcomparison of the group 1. In contrast, treatments of the HPMFs compoundin group 3 and group 4 resulted in the decreased expression levels ofPPARγ, C/EBPα, ACC, FAS and aP2 with comparison of the group 2.

According to FIG. 5, it shows that there is no significant difference inthe expression levels of p-AMPKα (Thr172) and p-AMPKβ (Ser108) betweenthe group 1 and the group 2. Comparing with the group 2, the expressionlevels of p-AMPKα (Thr172) and p-AMPKβ (Ser108) in the group 3 or thegroup 4 are increased and the expression levels of SREBP1c in the group3 or the group 4 are decreased. Moreover, the higher concentration ofthe HPMFs compound was treated, the more p-AMPKα (Thr172) and p-AMPKβ(Ser108) will be express. As shown in FIG. 6, the expression levels ofp-PI3K(Tyr508) and p-AKT(Ser473) in the group 2 are increased withcomparison of the group 1, and the expression levels of p-PI3K(Tyr508)and p-AKT(Ser473) in the group 3 are decreased with comparison of thegroup 2.

The results in FIG. 3 to FIG. 6 suggest that the HPMFs compound iscapable of suppressing the expression of transcription factor requiredfor adipocyte differentiation such as PPARγ, C/EBPα and their downstreamproteins including ACC, FAS and aP2. In addition, treatment of the HPMFscompound activates AMPK signaling pathway through phosphorylation onAMPKα (Thr172) and AMPKβ (Ser108). In contrast, treatment of the HPMFscompound suppresses expression level of PPARγ through reducing theprotein level of SREBP-1c. In addition, treatment of the HPMFs compoundsuppresses PI3K/AKT signaling pathway through suppresses thephosphorylation on PI3K (Tyr508) and AKT (Ser473). Therefore, it showsthat the effects of the HPMFs compound in controlling gene expressionprofile and modulating the molecular signaling pathway are capable ofpreventing the maturation of 3T3-L1 pre-adipocytes.

Example 6 The Cell Cycle Analysis of 3T3-L1 Pre-Adipocytes

The 3T3-L1 pre-adipocytes passaged in 24-wells plate were cultured withFBS-containing DMEM for 3 days. In the next step, the cells werecultured with fresh FBS-containing DMEM medium for 2 days. The day afterthe indicated culture procedure for 5 days was destined as “day 0”. Inthe following steps, the cells were grouped and treated with differentculture conditions, wherein the group 1 was the blank control, the group2 was the control group treated with DMI on day 2, the group 3 wastreated with DMI-containing medium and added 10 μg/mL of the HPMFscompound on day 2 and the group 4 was treated with DMI-containing mediumand added 20 μg/mL of the HPMFs compound on day 2.

The cultured cells in the each group were fixed for propidium iodide(hereinafter referred to as PI) staining on 18 and 24 hour. ThePI-stained cells in the each group were further analyzed by flowcytometry and software (ModFit LT) to determine the cells cycleprogression. The results of cell cycle analysis were showed in FIG. 7A,7B and table 1.

TABLE 1 Distribution of cell cycle progression of each group ondifferent time points Time Phases of cell cycle (%) points Groups G0/G1S G2/M 18 hours 1 76.72 ± 1.13 15.36 ± 3.27 7.60 ± 2.41 2 27.61 ± 1.6371.89 ± 1.65 0.50 ± 0.53 3 70.08 ± 1.64 29.64 ± 2.03 0.04 ± 0.05 4 83.66± 0.56 12.21 ± 0.27 5.22 ± 0.82 24 hours 1 77.05 ± 0.89 13.96 ± 0.278.59 ± 0.75 2 49.13 ± 1.35 15.40 ± 0.24 35.47 ± 1.12  3 30.67 ± 0.8848.87 ± 1.44 10.17 ± 0.19  4 82.59 ± 0.44 12.57 ± 1.04 4.84 ± 1.48

As shown in FIG. 7A, 7B and table 1, after treating for 18 hours, thereare 76.72% of the cultured 3T3-I1 pre-adipocytes in the group 1 intoG0/G1 phase and only 15.36% of the cultured 3T3-L1 pre-adipocytes in thegroup 1 progressing into S phase, 71.89% of the cultured 3T3-L1pre-adipocytes in the group 2 into S phase, 29.64% of the cultured3T3-L1 pre-adipocytes in the group 3 into S phase and 12.21% of thecultured 3T3-L1 pre-adipocytes in the group 4 into S phase. Furthermore,after treating for 24 hours, the cultured 3T3-L1 pre-adipocytes in thegroup 1 all retain in G0/G1 phase, 35.47% of the 3T3-L1 pre-adipocyte inthe group 2 progress into G2/M phase, 10.17% of the 3T3-L1 pre-adipocytein the group 3 are into G2/M phase and 4.84% of the 3T3-L1 pre-adipocytein the group 4 progress to G2/M phase.

According to the results in FIG. 7, it indicates that treatment of DMIon 3T3-L1 pre-adipocyte will induce mitotic clonal expansion, however,the HPMFs compound will decrease the mitotic clonal expansion and retainthe cells in G0/G1 phases to prevent post-mitotic adipocytedifferentiation and lipogenesis.

Example 7 Preparation of Obesity Mouse Model

The 4 weeks-old male C57BL/6 mice were divided into 4 groups and treatedwith different feeding conditions, wherein the group 1 was the blankcontrol fed with normal diet and water, the group 2 was fed withhigh-fat diet and water the group 3 was fed with high-fat diet, waterand 250 mg/Kg the HPMFs compound, and the group 4 was fed with high-fatdiet, water and 10 g/Kg the HPMFs compound. Feeding for 10 weeks, thebody weights of the mice in the each group were monitored every week asshown in FIG. 8 and the dietary intakes of the mice in the each groupswere statistically recorded as shown in FIG. 9. In addition, the grossviews of the mice in the each group were shown in FIG. 10A˜10D.

The results shown in FIG. 8 to FIG. 10 indicate that the dietary intakesrecorded from week 0 to week 10 are similar between the four groups.Observing the change of body weight in the each group, the mice of thegroup 1 are from 20.27 g to 28.31 g, the mice of the group 2 are from19.93 g to 35.11 g, the mice of the group 3 are from 19.15 g to 27.18 gand the mice of the group 4 are from 19.32 g to 27.18 g.

Consistent with the body weights, the mice in the group 2 revealsobviously larger body size than the mice in the group 1 afterconditional feeding for 10 weeks. The gross view of the mice in thegroup 3 or in the group 4 reveal smaller size than that in the group 2,furthermore, the gross view of the mice in the group 4 are similar thanthat in the group 1.

Therefore, it shows that feeding with high-fat diet is capable ofpreparing the mouse model of obesity. In addition, it can prevent thebody weight of obesity mouse to increase by oral feeding with the HPMFscompound. In other words, it suggests that the HPMFs compound is able toinhibit the increase of body weight without changing dietary intakes.

Example 8 Analyzing Weights of Internal Organs of the Mice in the EachGroup

The mice in the each group of example 7 were sacrificed for dissection.The weights of liver, kidney and spleen of the mice in the each groupwere measured and recorded in table 2.

TABLE 2 Weights of internal organs collected from the mice in the eachgroup Organ Group 1 Group 2 Group 3 Group 4 Liver 1.14 ± 0.17 1.47 ±0.07 1.30 ± 0.16 1.23 ± 0.08 Kidney 0.45 ± 0.02 0.54 ± 0.05 0.44 ± 0.050.40 ± 0.03 Spleen 0.06 ± 0.01 0.07 ± 0.01 0.07 ± 0.02 0.07 ± 0.01

In Table 2, the weights of liver and kidney in the group 2 increasing0.33 g and 0.06 g with the comparison of group 1, respectively. Itsuggests that the excessive accumulation of fat results in the increasedweight of the internal organs. Comparing with the group 2, the weightsof the liver and kidney in the group 3 decrease 0.17 g and 0.10 g,respectively and that in the group 4 decrease 0.24 g and 0.14 g,respectively.

According to the results in table 2, it shows that administering theHPMFs compound will inhibit the lipid accumulation in internal organsdecrease and the effect will be better with administering more the HPMFscompound. Furthermore, because the weights of the spleen between thefour groups are similar, it indicates that the HPMFs compound doesn'thave the cell toxicity.

Example 9 Analysis of the Internal Fat of Mice in the Each Group

After sacrificed the mice of the each group in example 7, the fat padssurrounding gonads, abdomen and intestine of the mice in the each groupwere be observed as shown were in FIG. 11 and FIG. 12, wherein FIGS. 11Ato 11D were sequentially the gross views of the fat pads surroundinggonad in the group 1 to 4, and FIG. 12A to 12D were sequentially thegross views of the abdominal fat pads in the group 1 to 4. Furthermore,measuring the weights of the fat pads surrounding gonads, abdomen andintestine collected from the mice in the each group, the results wereshown below:

The weights of the fat pads surrounding gonad, abdomen and intestine inthe group 1 were 0.60 g, 0.06 g and 0.34 g, respectively. The weights ofthe fat pads surrounding gonad, abdomen and intestine in the group 2were 1.65 g, 0.56 g and 0.67 g, respectively. The weights of the fatpads surrounding gonad, abdomen and intestine in the group 3 were 0.79g, 0.21 g and 0.39 g, respectively. The weights of the fat padssurrounding gonad, abdomen and intestine in the group 4 were 0.42 g,0.06 g and 0.27 g, respectively. These weights of the internal fat padsin the four groups were further statistical analysis and shown in FIG.13 to FIG. 15, wherein FIG. 13 shows the results of the weights of thefat pads surrounding gonad in the each group, FIG. 14 shows the resultsof the weights of the fat pads surrounding abdomen in the each group andFIG. 15 shows the results of the weights of the fat pads surroundingintestine in the each group.

According to FIG. 11 and FIG. 12, it suggests that the gross views ofthe fat pads surrounding gonad and abdomen in the group 2 are obviouslylarger than that in the group 1. Comparing with the group 2, the grossviews of the fat pads surrounding gonad and abdomen in the group 3 and 4were obviously reduced size.

Furthermore, the results in FIG. 13 and FIG. 14 show the increases of1.05 g, 0.50 g and 0.33 g on the fat pads surrounding gonad, abdomen andintestine of the group 2 with comparison of the group 1, respectively.And comparing with the group 2, the weights of the fat pads surroundinggonad, abdomen and intestine of the group 3 separately decreases 0.86 g,0.35 g and 0.26 g and that of the group 4 separately decreases 1.23 g,0.50 g and 0.38 g. The results in FIG. 11 to FIG. 15 suggest that theHPMFs compound is able to inhibit the growth of the adipose tissue witha dose dependent manner.

Example 10 Biochemistry Assays of the Serum Collected from the Mice inthe Each Group

After sacrificed the mice of the each group in example 7, the blood ofthe mice in the each group was collected. After centrifuge, the serumcollected from blood of the each group was used for biochemistry assaysto determine the concentrations of GOT, GPT, TG and T-cho in the serum.These measured biochemistry indexes are the typical indicators for liverfunctions shown in table 3.

TABLE 3 Contents of the biochemistry function of liver in the each groupGroup GOT (U/L) GPT (U/L) TG (mg/dl) T-cho (mg 1 77.40 ± 9.15  19.00 ±2.12  89.60 ± 28.61  80.60 ± 16.22 2 73.80 ± 29.02  29.80 ± 2.86^(#)117.20 ± 20.87 140.20 ± 15.30 3 78.80 ± 15.78 27.20 ± 3.96 111.40 ±13.31 130.40 ± 13.77 4 54.00 ± 13.69 25.60 ± 8.56  89.00 ± 21.42 119.00± 6.36 

The results in table 3 show that the increased concentrations of GPT, TGand T-cho in the serum of mice in the group 2 with comparison of thegroup 1. Moreover, the concentrations of GOT, GPT, TG and G-cho in theserum of the mice in the group 3 and the group 4 are decreased withcomparison of the group 2, respectively. Therefore, the results in table3 suggest that the HPMFs compound has ability to lower the risk of fattyliver and lipid accumulation with a dose dependent manner.

Example 11 Histological Analysis of Liver in the Each Group

After sacrificed the mice of the each group in example 7, the livercollected from the sacrificed mice in each groups was fixed withformalin. After the fixation, the liver tissue of the each group wasparaffin-embedded for histology section and analysis. The deparaffinedhistology sections of the four groups were subjected for hematoxylieneand eosin staining, respectively and shown in FIG. 16, wherein FIG. 16Ato 16D sequentially present in the group 1 to 4.

According to FIG. 16, it shows that the hepatocytes of the mice in thegroup 2 reveal the histopathology resulted from accumulation of largeamount of lipid-drops with comparison of the group 1. Comparing with thegroup 2, the group 3 can decrease the accumulation of oil-drops inhepatocytes. Furthermore, the group 4 not only decreases theaccumulation lipid-drops in hepatocytes, but also prevents theabnormality occurred on the histology of hepatocytes. Therefore, itindicates the effect of the HPMFs compound for improving the hepatichistology with a dose dependent manner.

The present invention discloses the effects of the HPMFs compound forinhibiting adipocyte differentiation and maturation, and preventingfatty liver and lowering lipid accumulation. Therefore, the HPMFscompound plays an important role in a pharmaceutical composition for thepurposes to suppress obesity and cure fatty liver. And based on theHPMFs compound extracted from the nature plants, it reveals no cellulartoxicity and the lower side effects to a subject. Therefore, the HPMFscompound is capable of applying as the dietary composition formanufacture of the functional foods to improve the obesity and improvingfatty liver.

The above-mentioned specification is only for detailed describing theexamples of the invention and shall not be construed as a limitation ofthe scope of the invention Thus, any modification or change withoutdeparting from the characteristics of the invention or any equivalentthereof shall be included in the scope of the invention defined in thefollowing claims.

1. A method of treating or preventing a disease selected from the groupconsisting of obesity, fatty liver, metabolic syndrome, insulinresistance syndrome, cardiovascular disease, hypertension andhyperlipidemia, comprising step A: taking a predetermined amount of anextract from a peel of citrus fruit; step B: dissolving the extract withalcohols and then adding hydrochloric acid to obtain a first mixture;step C: incubating the first mixture with heating circumfluence, andthen removing the alcohols, so as to obtain a second mixture; step D:extracting the second mixture by water and an organic solvent; step E:collecting an organic phase obtained with the organic solvent, and thenpurifying, so as to obtain a hydroxyl polymethoxylflavones (HPMFs)compound; and step F: administering an effective amount of a compositionto a subject, the composition including the hydroxylpolymethoxylflavones compound, a pharmaceutically acceptable saltthereof, or a mixture thereof.
 2. (canceled)
 3. The method of claim 1,wherein the citrus fruit is selected from the group consisting ofpomelo, mandarin orange, orange, kumquat and lemon.
 4. The method ofclaim 1, wherein the composition is a food composition.
 5. The method ofclaim 1, wherein the composition is a pharmaceutical composition. 6.(canceled)
 7. (canceled)